There is very little known about molecules secreted by protozoan parasites capable of communicating via receptors with host cells. The objective of this research proposal is to test the hypothesis that Leishmania major-derived secreted factors, possibly processed by the novel serine hydrolase oligopeptidase B, perform important functions in parasite to host cell Ca2+ signaling, infectivity and intracellular survival. Oligopeptidase B was first identified in this laboratory in Trypanosoma cruzi. It is a novel serine endopeptidase which plays an important role in generating a soluble Ca2+ signaling factor, produced by the infective stages of T. cruzi which interacts with receptors on mammalian cells and triggers intracellular Ca2+ signaling pathways. Gene deletion studies have recently demonstrated that infectivity of T. cruzi trypomastigotes is significantly decreased in the absence of oligopeptidase B, both in vitro and in vivo. A close homologue of the T. cruzi oligopeptidase B gene in L. major was recently cloned and sequenced in our laboratory. A less than 3kDa Ca2+ signaling factor specific for macrophages and epithelial cells was also detected in L. major soluble extracts. Specific aim 1 of this proposal involves generating a null mutant of the oligopeptidase B gene in L. major by targeted gene disruption. By doing so we can examine what effect this gene knockout has upon the parasite's ability to invade and survive within host cell macrophages in vitro and in vivo, and its role in the suppression of IL-12 production by L. major in macrophages. Specific aim 2 is designed to address the question of whether or not the less than 3kDa soluble Ca2+ signaling factor is still present in the L. major oligopeptidase B double null mutants, and whether the less than 3kDa factor is involved in IL- 12 regulation. Specific aim 3 involves further characterization of the macrophage signaling pathways responsible for the intracellular free Ca2+ mobilization, and fractionation techniques to help identify the less than 3kDa factor. These specific aims were designed so that important questions will be answered independently, the results of one aim not preventing the fulfillment of the others. This proposal constitutes the candidate's doctoral research project. The candidate will continue to expand her current knowledge and training as a Doctor of Veterinarian Medicine while acquiring training and research expertise in Parasitology, Cell and Molecular Biology and Biochemistry with the objective of developing into a successful and productive independent investigator.